UNDERSTANDING THE RELATIONSHIP BETWEEN STRUCTURE AND FUNCTION 
AT THE MOLECULAR AND CELLULAR LEVELS 
David A. Agard, Ph.D., Associate Investigator 
The research in Dr. Agard's laboratory continues 
to be devoted to structural studies of biological 
problems, in an effort to understand the fundamen- 
tal relationships between structure and function at 
the molecular and cellular levels. Four areas of in- 
vestigation are being pursued. 
Structural Basis of Enzyme Specificity 
One of the fundamental functions of an enzyme is 
to be specific, that is, to limit the number of sub- 
strates on which it can act. Dr. Agard and his co- 
workers have chosen a-lytic protease as an ideal 
model system to investigate structural and energetic 
aspects of enzyme specificity. They are combining 
x-ray crystallography, site-directed mutagenesis, ki- 
netics, and theoretical approaches in seeking to un- 
derstand the relationship between enzyme structure 
and function. 
Structural analysis has provided surprising in- 
sights into the mechanism of specificity and has 
indicated that flexibility plays a crucial role in se- 
lectivity. Current efforts involve mapping the ener- 
getics of protein flexibility through further muta- 
genesis and kinetic and crystallographic analyses. 
Recent experiments have shown that specificity can 
be altered by mutating residues that regulate flexi- 
bility. This is the first demonstration of a mechanism 
whereby substrate specificity can be modulated by 
residues distant from the binding pocket. 
A key test of one's understanding is to be able to 
predict the effect of mutations on substrate specific- 
ity. The Agard laboratory has brought this objective 
dramatically closer through a newly developed algo- 
rithm that combines the side-chain rotamer concept 
developed by Ponder and Richards with a complete 
force-field and solvent model. It has been possible 
to predict 40 experimental values of k^^JK^ for dif- 
ferent mutant-substrate combinations for a-lytic 
protease and 1 00 values for subtilisin with excep- 
tional accuracy: an average error of <0.7 kcal/mol, 
or a factor of ~ 3 in k^^JK^. This approach has per- 
mitted de novo design of an enzyme with a new 
pattern of specificity and has proved very effective 
for modeling the structure of an unknown protein 
based on a homologous structure. Current efforts 
center on the use of this free-energy function to re- 
fine the conformation of model-built substrates. 
This is a key step toward practical, rational drug 
design. 
Pro Regions as a New Class of Molecular 
Chaperones 
a-Lytic protease is synthesized as a prepro- 
enzyme. Experiments in the Agard laboratory have 
demonstrated that the l66-amino acid pro region is 
absolutely required for the proper folding of the 
198-amino acid protease domain, either in vivo or 
in vitro. Significantly, the covalent linkage between 
the pro region and the protease domain is not re- 
quired for function. In principle, the pro region 
could function by reducing the rate of off-pathway 
folding reactions (as suggested for the "classical" 
molecular chaperonins) or by increasing the rate of 
a limiting on-pathway reaction. The laboratory has 
recently found that the pro region is a potent inhibi- 
tor of the mature protease. This suggests that the pro 
region directly facilitates an on-pathway reaction 
and that the rate-limiting folding transition state has 
a native-like conformation. 
Remarkably, the Agard group has been able to trap 
a stable folding intermediate under nondenaturing 
conditions at physiological pH. This provides a 
unique opportunity to examine the folding pro- 
cesses in detail, structurally and functionally. The 
intermediate rapidly folds to the native state upon 
addition of the pro region. Detailed kinetic analysis 
indicates that the pro region promotes folding by 
directly speeding the rate-limiting step on the fold- 
ing pathway by >10^. By mutagenesis, it appears 
that a residue in the pro region that plays a "cata- 
lytic" role has been isolated. This is a major step 
toward understanding the mechanism of folding ca- 
talysis. Recent work has also revealed that the inter- 
mediate has many features of a molten globule: ex- 
panded radius, considerable secondary structure, 
and little or no tertiary structure. Current work fo- 
cuses on further structural and functional character- 
ization of the folding intermediate and of the pro 
region complexed with native protease, using genet- 
ics, spectroscopy, nuclear magnetic resonance 
(NMR), and x-ray crystallography. This study has 
also provided new insights into the mechanism of 
secretion of proteins through the outer membrane 
of Escherichia coli. 
Three-Dimensional Analysis 
of Chromosome Structure 
The Agard laboratory is engaged in a close collabo- 
ration with Dr. John Sedat (HHMI, University of Cali- 
STRUCTURAL BIOLOGY 46 1 
